This research focuses on oxidative modification of proteins. The resulting covalent modifications have been implicated in important physiologic and pathologic processes. Determination of the actual roles of oxidative modification in these processes requires the identification of specific proteins which are susceptible to modification and the mapping of the sites of modification in those proteins. During this year, heavy emphasis was again placed on the development of novel methods for the detection of modified proteins and quantitation of those modifications. In particular, we have pursued mass spectrometric methods for the identification of oxidatively modified proteins. The modified proteins are detected by a Western blot for carbonyl groups and then purified by classical techniques, or preferably, by 1-D and 2-D gel purifications. The protein bands are then excised, digested by proteases, and subjected to mass spectrometry by either electrospray or MALDI techniques. The sensitivity of this approach has allowed identification of some of the oxidatively modified proteins of interest, but for others it was not yet sufficient for confident identification. The same methodology, coupled with automated Edman sequencing, has been applied to a major collaborative effort with the Laboratory of Cell Signaling. Our goal is to identify the proteins in brain which are sensitive to oxidation of specific cysteine residues. Over twenty such proteins have been unambiguously identified thus far. Having identified proteins which are sensitive to various oxidative modifications, we can now study the physiologic and pathologic effects of the modifications.